Anti cd14 bv711

PBMCs and other cells were stained with anti-human mAbs: anti-CD3 FITC, anti-CD4 BV605, anti-CD8 BV421, anti-CD14 BV711, anti-CD19 BV605, anti-CD1d APC, anti-HLA-DR BV421, IFN-γ PE, and IL-4 PE (all from BD Biosciences, Vienna, Austria). APC-labeled human CD1d tetramers loaded with the α-GalCer analogue PBS-57 were obtained from the MHC Tetramer Core Facility (Emory University Vaccine Center, Atlanta, GA). Anti-human ADRB2 (AbD Serotec, Oxford, UK) was coupled with alexa fluor647 fluorochrome by using a protein labeling kit (Life Technologies, Paisley, UK). Viability was assessed with Zombie Nir viability dye (BioLegend, London, UK) or with fixable viability dye eFluor506 (eBiosciences, Vienna, Austria). The corresponding isotype control mAbs were used to assess staining specificity.

Endotoxin tolerance was induced as previously described: 2.5x10⁶ PBMCs/mL were incubated in the presence or absence of 100 ng/mL LPS for 48 h. Cells from each group were then washed, split in different tubes, and incubated with 2 mM 2-deoxy-D-Glucose (2-DG; Cayman Chemical, Ann Arbor, MI) to limit glycolysis, 1 µM Oligomycin A (Cayman Chemical) to inhibit OXPHOS, or 100 µM 6-Aminonicotinamide (6-AN; Cayman Chemical) to inhibit the pentose phosphate pathway (PPP). To evaluate cytokine production, 100 ng/mL LPS challenge were added to the cells 1h after the metabolic modulators. After 24 h incubation, supernatants were collected and stored at -80° C for further measurement of TNF-α and IL-6 using a Cytometric Bead Array (CBA; BD Biosciences) following the manufacturer’s instructions. To measure the phagocytic capacity, endotoxin-tolerant and control PBMCs were incubated for 22 h with the metabolic modulators before a 2.5 h incubation with 0.5mg/mL pHrodo Green E. coli BioParticles (Thermo Fisher Scientific). Cells were stained with anti-CD14-BV711 (BD Biosciences) for monocyte identification and pHrodo green median fluorescence intensity was determined by flow cytometry.